Cholangiocytes express an isoform of soluble adenylyl cyclase that is N‐linked glycosylated and secreted in extracellular vesicles

Soluble adenylyl cyclase (sAC)‐derived cAMP regulates various cellular processes; however, the regulatory landscape mediating sAC protein levels remains underexplored. We consistently observed a 85 kD (sAC85) or 75 kD (sAC75) sAC protein band under glucose‐sufficient or glucose‐deprived states, resp...

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Published in:Traffic (Copenhagen, Denmark) Denmark), 2023-09, Vol.24 (9), p.413-430
Main Authors: Go, Simei, Li, Hang Lam, Chang, Jung‐Chin, Verhoeven, Arthur J., Elferink, Ronald P. J. Oude
Format: Article
Language:English
Subjects:
Adenylate cyclase
adenylyl cyclases
Adenylyl Cyclases - metabolism
Deglycosylation
Epithelial Cells - metabolism
Extracellular vesicles
Extracellular Vesicles - metabolism
Glucose
Glucose - metabolism
glycosylation
Immunoblotting
Intracellular
Protein Isoforms
protein transport
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Summary:Soluble adenylyl cyclase (sAC)‐derived cAMP regulates various cellular processes; however, the regulatory landscape mediating sAC protein levels remains underexplored. We consistently observed a 85 kD (sAC85) or 75 kD (sAC75) sAC protein band under glucose‐sufficient or glucose‐deprived states, respectively, in H69 cholangiocytes by immunoblotting. Deglycosylation by PNGase‐F demonstrated that both sAC75 and sAC85 are N‐linked glycosylated proteins with the same polypeptide backbone. Deglycosylation with Endo‐H further revealed that sAC75 and sAC85 carry distinct sugar chains. We observed release of N‐linked glycosylated sAC (sACEV) in extracellular vesicles under conditions that support intracellular sAC85 (glucose‐sufficient) as opposed to sAC75 (glucose‐deprived) conditions. Consistently, disrupting the vesicular machinery affects the maturation of intracellular sAC and inhibits the release of sACEV into extracellular vesicles. The intracellular turnover of sAC85 is extremely short (t1/2 ~30 min) and release of sACEV in the medium was detected within 3 h. Our observations support the maturation and trafficking in cholangiocytes of an N‐linked glycosylated sAC isoform that is rapidly released into extracellular vesicles. We demonstrate the glucose‐dependent trafficking and maturation of a novel membrane‐enclosed N‐linked glycosylated isoform of soluble adenylyl cyclase that is released in extracellular vesicles.
ISSN:1398-9219
1600-0854
DOI:10.1111/tra.12904